Genome mining yields putative disease-associated ROMK variants with distinct defects.

Area of Science:

• Genetics
• Molecular Biology
• Nephrology

Background:

• Bartter syndrome is a rare genetic kidney disorder affecting electrolyte reabsorption, potentially leading to fatal dehydration and electrolyte imbalances.
• Bartter syndrome type II is caused by mutations in the KCNJ1 gene, encoding the renal outer medullary potassium channel (ROMK), with many mutations' molecular defects remaining uncharacterized.
• Some known mutations disrupt ROMK protein folding, leading to its degradation via the ER-associated degradation (ERAD) pathway.

Purpose of the Study:

• To identify novel, uncharacterized human KCNJ1 variants causing Bartter syndrome type II by developing and applying a computational and experimental pipeline.
• To elucidate the molecular mechanisms underlying ROMK dysfunction caused by identified mutations.
• To establish a framework for identifying disease-associated alleles in other potassium channels.

Main Methods:

• Utilized UK Biobank, NIH TOPMed, and ClinVar databases to identify KCNJ1 variants.
• Employed a computational platform and Rhapsody algorithm to analyze phenotypic data and predict mutation pathogenicity.
• Conducted yeast screens for ROMK function, analyzed ROMK biogenesis in yeast and human cells, and performed electrophysiology in X. laevis oocytes.

Main Results:

• Identified four previously uncharacterized KCNJ1 mutations associated with Bartter syndrome type II.
• The G228E mutation destabilized ROMK, targeting it for ERAD and reducing cell surface expression.
• The T300R mutation was ERAD-resistant but impaired ROMK channel activity, as shown by electrophysiology.

Conclusions:

• The developed computational and experimental pipeline effectively identifies disease-causing KCNJ1 variants and characterizes their molecular defects.
• The findings deepen the understanding of ROMK structure-function relationships and provide insights into Bartter syndrome type II pathogenesis.
• This approach can be extended to discover disease-associated mutations in other potassium channels, advancing precision medicine.